Many studies have identified oncogenes and tumor suppressor genes as markers of cellular transformation in several tissue types, such as colon, pancreas and lung, whereas comparable studies in breast cancer have met with limited success (West et al., 2001, Proc. Natl. Acad. Sci. USA, 98, 11462). This reflects the difficulty in finding genetic and epigenetic alterations in a significant proportion of breast cancers, and also underscores the phenotypic heterogeneity of breast cancer. The identification of molecular targets for early diagnosis of breast cancer could lead to improved diagnosis and treatment based on a molecular diagnosis.
Most mammary carcinomas contain estrogen receptors (ER), which are important factors for diagnosis and prognosis of breast cancer, and for determining therapeutic choices (Osborne, 1998, Breast Cancer Res. Treat., 51, 227). Estrogens are direct mitogens for hormone-responsive human breast cancer cells, where they promote cell cycle progression and induce the transcriptional activation of “immediate early” and cyclin genes. The estrogen receptor alpha (ER-α) and its ligand (17β-estradiol) play a crucial role in normal breast development, and have also been linked to mammary carcinogenesis and clinical outcome in breast cancer patients. However, up to one third of breast cancers lack ER-α at the time of diagnosis, and a fraction of breast cancers that are initially ER-α-positive lose ER during tumor progression (Hortobagyi, 1998, New Engl. J. Med., 339, 974). In a significant fraction of breast cancers, the absence of ER-α gene expression has been associated with the aberrant methylation of its CpG islands (Hortobagyi, 1998; Weigel and Coninck, 1993, Cancer Res., 53, 3472).
There is abundant evidence that the structure and chemical composition of chromatin directly affects gene expression. Histones are the primary structural components of chromatin. The nucleosome is the basic repeating unit of chromatin; further compaction of nucleosomes, with the aid of the histone H1 and other non-histone proteins, leads to a condensed chromatin state (Hayes and Hansen, 2001, Curr. Opin. Genet. Dev., 11, 124). The chromatin is thus made inaccessible to the transcriptional machinery, resulting in gene silencing.
Chromatin structure and function are controlled, at least in part, through post-translational modifications of nucleosomal histones. The core histone tails are susceptible to a variety of covalent modifications, including acetylation, methylation, phosphorylation and ubiquitination. Different studies collectively support the “histone code hypothesis” of histone modification (Strahl and Allis, 2000, Nature, 403, 41), which suggests that the presence of a given modification on histone tails may dictate or prevent the presence of a second modification elsewhere on the same histone. Histone modifications may therefore serve as marks for the recruitment of different proteins or protein complexes, which regulate chromatin functions such as gene expression.
DNA methylation is also important for transcriptional silencing. Therefore, it has been proposed that DNA methylation and histone deacetylation might work together to establish a repressive chromatin environment and silence gene expression (Cameron et al., 1999, Nat. Genet., 21, 103). For example, the formation of transcriptional repression complexes such as DNA methyltransferase 1 (DNMT1)/histone deacetylase (HDAC) is emerging as an important mechanism in gene expression regulation (Grunstein, 1997, Nature, 389, 349; Struhl, 1998, Genes & Dev. 12, 599; Lin et al., 1998, Nature, 391, 8311; Laird and Jaenisch, 1996, Annu. Rev. Genet. 30, 441). Aberrant recruitment of HDAC activity has also been associated with the development of certain human cancers (Nan et al., 1998, Nature, 393, 386) and changes in the patterns of CpG-methylation appear to be an intrinsic feature of human malignancy (Jones et al., 1998, Nat. Genet., 19, 187). However, the mechanisms of gene silencing by methylation remain poorly understood. Recent studies suggest that histone methylation, similar to histone deacetylation, might function in concert with DNA methylation (Bird and Wolffe, 1999, Cell, 99, 451), or that histone methylation on lysines by the histone methyl transferase SUV39H1 is important for transcriptional silencing. A specific chromatin structure involving methylated histones may also be necessary for DNA methylation to occur (Ng and Bird, 1999, Curr. Opin. Genet. Dev., 9, 158).
Several mechanisms have been proposed to account for transcriptional repression by the Rb proteins (Magnaghi-Jaulin et al., 1998, Nature, 391, 601; Dunaief et al., 1994, Cell, 79, 119, Trouche et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 11268). Some of the proposed models stress the importance of chromatin structure in regulating transcriptional activity. Active repression by Rb family members could involve a mechanism by which condensed chromatin structure is enhanced through histone deacetylation and methylation. Rb proteins have been shown to repress E2F-dependent transcription by recruiting HDAC1/2 (Iavarone and Massague, 1999, Mol. Cell Biol., 19, 916; Stiegler et al., 1998, Cancer Res., 58, 5049). Recent data show that pRb2/p130 and p107 are able to interact physically with HDAC1 through the A/B pocket domains (Magnaghi-Jaulin et al., 1998; Iavarone and Massague, 1999; Ferreira et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 10493).
Repression of E2F-responsive promoters in quiescent cells is associated with E2F-4 and pRb2/p130 recruitment and low histone acetylation levels. Recently, different studies have shown that SUV39H1 is involved in transcriptional repression by the retinoblastoma protein Rb1/p105 (Vandel et al., 2001, Mol. Cell. Biol., 21, 6484).
Chromatin inactivation mediated by histone deacetylation and DNA methylation are critical components of ER-α silencing in human breast cancer cells. In vitro studies have shown that DNMT1 interacts physically with either HDAC1 or 2, and that co-treatment with DNMT1 and HDAC inhibitors can synergistically induce ER-α gene expression in ER-α-negative breast cancer cells (Rountree et al., 2000, Nat. Genet., 25, 269; Robertson et al., 2000, Nat. Genet., 25, 338; Yang et al., 2001, Cancer Res., 60, 6890). However, the molecular factors which promote DNMT1 and HDAC interaction and otherwise regulate the ER-α gene expression have not heretofore been identified.
The ability to identify breast cancer patients with more aggressive diseases is crucial to an accurate prognosis and the planning of an adequate treatment. For example, those breast cancers which are estrogen-receptor negative (also called estrogen-insensitive breast cancers) have a higher malignant potential. Typically, metastatic potential is determined by considering a range of pathologic tumor features, including histologic type, grade of differentiation, depth of invasion, and extent of lymph nodal metastases. Unfortunately, these factors do not always allow a sufficiently accurate determination of metastatic potential of breast cancer. Such parameters also have questionable reproducibility. Estrogen-receptor negative breast cancers are also less susceptible to treatment with anticancer drugs such as tamoxifen.
What is needed, therefore, is a method of detecting and regulating the molecular factors which control ER-α gene expression, particularly in estrogen receptor-negative breast cancer cells. The detection and regulation of such factors would allow estrogen-insensitive breast cancer cells to be identified, so that an accurate prognosis can be obtained and an appropriate course of treatment administered. Also, detecting and regulating the molecular factors which control ER-α gene expression would allow estrogen-insensitive cells to be converted to estrogen-sensitive cells, which are generally more susceptible to current anti-cancer treatments.